Summary A novel coronavirus (CoV), SARS-CoV-2, is causing global pandemics with growing numbers of daily cases and deaths. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2002-2003 SARS epidemic, and less closely related to MERS-CoV, which causes a high mortality rate in infected patients. No therapeutic agents or vaccines have been approved to control the infections of these CoVs in humans, calling for immediate efforts to develop effective countermeasures. The CoV spike (S) proteins are important targets for therapeutics. They guide virus entry into host cells by binding to a host receptor through their S1 subunits and fusing the viral and host cell membranes through their S2 subunits. The S1 subunits contain a receptor-binding domain (RBD). Both RBD and S2 region of S protein can elicit neutralizing antibodies. The RBD is the major target to induce potent neutralizing antibodies, but it diverges among different CoVs, whereas the S2 region of S protein is more conserved among different CoVs. Nanobodies (Nbs) are single-domain antibodies derived from camelid antibodies. They are emerging as novel therapeutic agents, numbers of which have been approved or tested in clinical trials to prevent and treat other human diseases. Nbs possess many unique advantages as therapeutic agents: they have high physical and chemical stabilities, excellent tissue penetration capability (superior pharmacokinetics), easy expression with great production yields, and robustness for storage and transportation. Moreover, Nbs can potentially recognize epitopes (e.g. hidden or partially hidden epitopes) that are not accessible to conventional antibodies. In this proposal, we will design and develop Nbs as therapeutic agents against SARS-CoV-2 and other pathogenic human CoVs using phage display and structural biology as guiding tools. We propose to develop highly efficacious Nbs against SARS- CoV-2 to stop the current COVID-19 pandemic and also broad-spectrum neutralizing Nbs targeting future CoV infections. We have established Nb libraries and identified several neutralizing Nbs targeting the RBDs of SARS-CoV and MERS-CoV, providing a solid foundation for the proposed studies. In our previous work, we extensively characterized the structures and functions of MERS-CoV and SARS-CoV S proteins, and we have recently characterized the RBD of SARS-CoV-2 and solved its structure in complex with viral receptor, paving the way for rapid screening and identification of SARS-CoV-2 S protein-based Nbs. The specific aims of this proposal are to: 1) develop highly efficacious Nbs targeting SARS-CoV-2 S (RBD/S2) protein; develop broad- spectrum Nbs against CoV infections, 2) characterize these CoV S-targeting Nbs, and 3) evaluate in vivo efficacy of these Nbs against SARS-CoV-2 and other CoV infections. Overall, this proposal will develop highly efficacious Nbs targeting SARS-CoV-2 S protein, aiming to stop the current COVID-19 pandemic. It will also develop broad-spectrum Nbs targeting future CoV infections. This proposal has important implications for combating pathogenic coronaviruses and neutralizing their threat to human health.